Hybrid maize plant and seed

ABSTRACT

This invention relates to a hybrid maze plant, designated as X1069G, produced by crossing two Pioneer Hi-Bred International., Inc inbred maize lines GE535769 and GE515721. This invention thus relates to the hybrid seed X1069G, the hybrid plant produced from the seed, and variants and trivial modifications of hybrid X1069G. This invention also relates to methods for producing a X1069G hybrid maize plant containing genetic material for one or more desirable traits and to the maize plant produced by that method. This invention further relates to methods for making maize lines produced from hybrid maize line X1069G.

FIELD OF THE INVENTION

This invention is in the field of maize breeding, specifically relatingto hybrid maize designated X1069G.

BACKGROUND OF THE INVENTION Plant Breeding

Field crops are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinated if pollen fromone flower is transferred to the same or another flower of the sameplant. A plant is cross-pollinated if the pollen comes from a flower ona different plant

Plants that have been self-pollinated and selected for type for manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny. A cross between twodifferent homozygous lines produces a uniform population of hybridplants that may be heterozygous for many gene loci. A cross of twoplants each heterozygous at a number of gene loci will produce apopulation of hybrid plants that differ genetically and will not beuniform.

Maize (Zea mays L.), often referred to as corn in the United States, canbe bred by both self-pollination and cross-pollination techniques. Maizehas separate male and female flowers on the same plant, located on thetassel and the ear, respectively. Natural pollination occurs in maizewhen wind blows pollen from the tassels to the silks that protrude fromthe tops of the ears.

The development of a hybrid maize variety in a maize plant breedingprogram involves three steps: (1) the selection of plants from variousgermplasm pools for initial breeding crosses; (2) the selfing of theselected plants from the breeding crosses for several generations toproduce a series of inbred lines, which, although different from eachother, breed true and are highly uniform; and (3) crossing the selectedinbred lines with unrelated inbred lines to produce the hybrid progeny(F1). During the inbreeding process in maize, the vigor of the linesdecreases. Vigor is restored when two different inbred lines are crossedto produce the hybrid progeny (F1). An important consequence of thehomozygosity and homogeneity of the inbred lines is that the hybridcreated by crossing a defined pair of inbreds will always be the same.Once the inbreds that create a superior hybrid have been identified, acontinual supply of the hybrid seed can be produced using these inbredparents and the hybrid corn plants can then be generated from thishybrid seed supply.

Large scale commercial maize hybrid production, as it is practicedtoday, requires the use of some form of male sterility system whichcontrols or inactivates male fertility. A reliable method of controllingmale fertility in plants also offers the opportunity for improved plantbreeding. This is especially true for development of maize hybrids,which relies upon some sort of male sterility system. There are severaloptions for controlling male fertility available to breeders, such as:manual or mechanical emasculation (or detasseling), cytoplasmic malesterility, genetic male sterility, gametocides and the like.

Hybrid maize seed is typically produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twoinbred varieties of maize are planted in a field, and the pollen-bearingtassels are removed from one of the inbreds (female) prior to pollenshed. Providing that there is sufficient isolation from sources offoreign maize pollen, the ears of the detasseled inbred will befertilized only from the other inbred (male), and the resulting seed istherefore hybrid and will form hybrid plants.

The laborious, and occasionally unreliable, detasseling process can beavoided by using cytoplasmic male-sterile (CMS) inbreds. Plants of a CMSinbred are male sterile as a result of factors resulting from thecytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent inmaize plants, since only the female provides cytoplasm to the fertilizedseed. CMS plants are fertilized with pollen from another inbred that isnot male sterile. Pollen from the second inbred may or may notcontribute genes that make the hybrid plants male-fertile. Usually seedfrom detasseled fertile maize and CMS produced seed of the same hybridare blended to insure that adequate pollen loads are available forfertilization when the hybrid plants are grown.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed In U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Theseand all patents referred to are incorporated by reference. In additionto these methods, Albertsen et al., of Pioneer Hi-Bred, U.S. patentapplication Ser. No. 5,432,068, have developed a system of nuclear malesterility which includes: identifying a gene which is critical to malefertility; silencing this native gene which is critical to malefertility; removing the native promoter from the essential malefertility gene and replacing it with an inducible promoter; insertingthis genetically engineered gene back into the plant; and thus creatinga plant that is male sterile because the inducible promoter is not “on”resulting in the male fertility gene not being transcribed. Fertility isrestored by inducing, or turning “on”, the promoter, which in turnallows the gene that confers male fertility to be transcribed.

There are many other methods of conferring genetic male sterility in theart, each with its own benefits and drawbacks. These methods use avariety of approaches such as delivering into the plant a gene encodinga cytotoxic substance associated with a male tissue specific promoter oran antisense system in which a gene critical to fertility is identifiedand an antisense to that gene is inserted in the plant (see:Fabinjanski, et al. EPO 89/3010153.8 publication no. 329,308 and PCTapplication PCT/CA90/00037 published as WO 90/08828).

Another system useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see Carlson, Glenn R., U.S. Pat. No.: 4,936,904). Applicationof the gametocide, timing of the application and genotype specificityoften limit the usefulness of the approach.

The use of male sterile inbreds is but one factor in the production ofmaize hybrids. The development of maize hybrids in a maize plantbreeding program requires, in general, the development of homozygousinbred lines, the crossing of these lines, and the evaluation of thecrosses. Maize plant breeding programs combine the genetic backgroundsfrom two or more inbred lines or various other broad-based sources Into.breeding pools from which new inbred lines are developed by selfing andselection of desired phenotypes. Hybrids also can be used as a source ofplant breeding material or as source populations from which to developor derive new maize lines. Plant breeding techniques known in the artand used in a maize plant breeding program include, but are not limitedto, recurrent selection backcrossing, pedigree breeding, restrictionlength polymorphism enhanced selection, genetic marker enhancedselection and transformation. The inbred lines derived from hybrids canbe developed using said methods of breeding such as pedigree breedingand recurrent selection. New inbreds are crossed with other inbred linesand the hybrids from these crosses are evaluated to determine which ofthose have commercial potential.

Recurrent selection breeding, backcrossing for example, can be used toimprove inbred lines and a hybrid which is made using those inbreds.Backcrossing can be used to transfer a specific desirable trait from oneinbred or source to an inbred that lacks that trait. This can beaccomplished, for example, by first crossing a superior inbred(recurrent parent) to a donor inbred (non-recurrent parent), thatcarries the appropriate gene(s) for the trait in question. The progenyof this cross is then mated back to the superior recurrent parentfollowed by selection in the resultant progeny for the desired trait tobe transferred from the non-recurrent parent. After five or morebackcross generations with selection for the desired trait and for thegermnplasm inherited from the recurrent parent, the progeny will behomozygous for loci controlling the characteristic being transferred,but will be like the superior parent for essentially all other genes.The last backcross generation is then selfed to give pure breedingprogeny for the gene(s) being transferred. A hybrid developed frominbreds containing the transferred gene(s) is essentially the same as ahybrid developed from the same inbreds without the transferred gene(s).

Another increasingly popular form of commercial hybrid productioninvolves the use of a mixture of male sterile hybrid seed and malepollinator seed. When planted, the resulting male sterile hybrid plantsare pollinated by the pollinator plants. This method is primarily usedto produce grain with enhanced quality grain traits, such as high oil,because desired quality grain traits expressed in the pollinator willalso be expressed in the grain produced on the male sterile hybridplant. In this method the desired quality grain trait does not have tobe incorporated by lengthy procedures such as recurrent backcrossselection into an inbred parent line. One use of this method isdescribed U.S. Pat. Nos. 5,704,160 and 5,706,603.

There are many important factors to be considered in the art of plantbreeding, such as the ability to recognize important morphological andphysiological characteristics, the ability to design evaluationtechniques for genotypic and phenotypic traits of interest, and theability to search out and exploit the genes for the desired traits innew or improved combinations.

The objective of commercial maize hybrid line development resulting froma maize plant breeding program is to develop new inbred lines to producehybrids that combine to produce high grain yields and superior agronomicperformance. The primary trait breeders seek is yield. However, manyother major agronomic traits are of importance in hybrid combination andhave an impact on yield or otherwise provide superior performance inhybrid combinations. Such traits include percent grain moisture atharvest, relative maturity, resistance to stalk breakage, resistance toroot lodging, grain quality, and disease and insect resistance. Inaddition, the lines per se must have acceptable performance for parentaltraits such as seed yields, kernel sizes, pollen production, all ofwhich affect ability to provide parental lines in sufficient quantityand quality for hybridization. These traits have been shown to be undergenetic control and many if not all of the traits are affected bymultiple genes.

Pedigree Breeding

The pedigree method of breeding is the mostly widely used methodologyfor new hybrid line development.

In general terms this procedure consists of crossing two inbred lines toproduce the non-segregating F1 generation, and self pollination of theF1 generation to produce the F2 generation that segregates for allfactors for which the inbred parents differ. An example of this processis set forth below. Variations of this generalized pedigree method areused, but all these variations produce a segregating generation whichcontains a range of variation for the traits of interest.

EXAMPLE 1 Hypothetical Example of Pedigree Breeding Program

Consider a cross between two inbred lines that differ for alleles at sixloci.

The parental genotypes are:

Parent 1 AbCdeF/AbCdeF Parent 2 aBcDEf/aBcDEfthe F1 from a cross between these two parents is:

F1 AbCdeF/aBcDEfSelfing F1 will produce an F2 generation including the followinggenotypes:

-   -   ABcDEf/abCdeF    -   ABcDef/abCdEF    -   ABcDef/abCdeF

The number of genotypes in the F2 is 36 for six segregating loci (729)and will produce (26)-2 possible new inbreds, (62 for six segregatingloci).

Each inbred parent which is used in breeding crosses represents a uniquecombination of genes, and the combined effects of the genes define theperformance of the inbred and its performance in hybrid combination.There is published evidence (Smith, O. S., J. S. C. Smith, S. L. Bowen,R. A. Tenborg and S. J. Wall, TAG 80:833840 (1990)) that each of thelines are different and can be uniquely identified on the basis ofgenetically-controlled molecular markers.

It has been shown (Hallauer, Amel R. and Miranda, J. B. Of. QuantitativeGenetics in Maize Breeding, Iowa State University Press, Ames Iowa,1981) that most traits of economic value in maize are under the geneticcontrol of multiple genetic loci, and that there are a large number ofunique combinations of these genes present in elite maize germplasm. Ifnot, genetic progress using elite inbred lines would no longer bepossible. Studies by Duvick and Russell (Duvick, D. N., Mavdica37:69-79, (1992); Russell, W. A., Maydica XXIX:375-390 (1983)) haveshown that over the last 50 years the rate of genetic progress incommercial hybrids has been between one and two percent per year.

The number of genes affecting the trait of primary economic importancein maize, grain yield, has been estimated to be in the range of 10-1000.Inbred lines which are used as parents for breeding crosses differ inthe number and combination of these genes. These factors make the plantbreeder's task more difficult. Compounding this is evidence that no oneline contains the favorable allele at all loci, and that differentalleles have different economic values depending on the geneticbackground and field environment in which the hybrid is grown. Fiftyyears of breeding experience suggests that there are many genesaffecting grain yield and each of these has a relatively small effect onthis trait. The effects are small compared to breeders' ability tomeasure grain yield differences in evaluation trials. Therefore, theparents of the breeding cross must lit differ at several of these lociso that the genetic differences in the progeny will be large enough thatbreeders can develop a line that increases the economic worth of itshybrids over that of hybrids made with either parent.

If the number of loci segregating in a cross between two inbred lines isn, the number of unique genotypes in the F2 generation is 3n and thenumber of unique inbred lines from this cross is {(2n)−2}. Only a verylimited number of these combinations are useful. Only about 1 in 10,000of the progeny from F2's are commercially useful.

By way of example, if it is assumed that the number of segregating lociin F-2 is somewhere between 20 and 50, and that each parent is fixed forhalf the favorable alleles, it is then possible to calculate theapproximate probabilities of finding an inbred that has the favorableallele at {(n/2)+m} loci, where n/2 is the number of favorable allelesin each of the parents and m is the number of additional favorablealleles in the new inbred. See Example 2 below. The number m is assumedto be greater than three because each allele has so small an effect thatevaluation techniques are not sensitive enough to detect differences dueto three or less favorable alleles. The probabilities in Example 2 areon the order of 10-5 or smaller and they are the probabilities that atleast one genotype with (n/2)=m favorable alleles will exist.

To put this in perspective, the number of plants grown on 60 millionacres (approximate United States corn acreage) at 25,000 plants/acre is1.5×1012.

EXAMPLE 2 Probability of Finding an Inbred with m of n Favorable Alleles

Assume each parent has n/2 of the favorable alleles and only ½ of thecombinations of loci are economically useful.

No. of No. of favorable No. additional segregating alleles in Parentsfavorable alleles Probability that loci (n) (n/2) in new inbred genotypeoccurs* 20 10 14 3 × 10-5 24 12 16 2 × 10-5 28 14 18 1 × 10-5 32 16 20 8× 10-6 36 18 22 5 × 10-6 40 20 24 3 × 10-6 44 22 26 2 × 10-6 48 24 28 1× 10-6 *Probability that a useful combination exists, does not includethe probability of identifying this combination if it does exist.

The possibility of having a usably high probability of being able toidentify this genotype based on replicated field testing would be mostlikely smaller than this, and is a function of how large a population ofgenotypes is tested and how testing resources are allocated in thetesting program.

SUMMARY OF THE INVENTION

According to the invention, there is provided a hybrid maize plantdesignated as X1069G, produced by crossing two Pioneer Hi-BredInternational., Inc. proprietary inbred maize lines GE535769 andGE515721. These lines, deposited with the American Type CultureCollection, (ATCC), Manassas, Va. 20110, have accession number PTA-5522for G535769 and accession number PTA-1306 for GE515721. This inventionthus relates to the hybrid seed X1069G, the hybrid plant produced fromthe seed, and variants, mutants and trivial modifications of hybridX1069G. This invention also relates to methods for producing a maizeplant containing in its genetic material one or more transgenes and tothe transgenic maize plants produced by that method. This inventionfurther relates to methods for producing maize lines derived from hybridmaize line X1069G and to the maize lines derived by the use of thosemethods. This hybrid maize plant is characterized by outstanding yieldpotential with solid agronomic strengths and a good disease resistancepackage that provides a broad area of adaptation.

DEFINITIONS

In the description and examples that follow, a number of terms are usedherein. In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided. NOTE: ABS is in absolute termsand %MN is percent of the mean for the experiments in which the inbredor hybrid was grown. These designators will follow the descriptors todenote how the values are to be Interpreted. Below are the descriptorsused in the data tables included herein.

ABTSTK=ARTIFICIAL BRITTLE STALK. A count of the number of “snapped”plants per plot following machine snapping. A snapped plant has itsstalk completely snapped at a node between the base of the plant and thenode above the ear. Expressed as percent of plants that did not snap.

ADF=PERCENT ACID DETERGENT FIBER. The percent of dry matter that is aciddetergent fiber in chopped whole plant forage.

ANT ROT=ANTHRACNOSE STALK ROT (Colletotrichum graminicola). A 1 to 9visual rating indicating the resistance to Anthracnose Stalk Rot. Ahigher score indicates a higher resistance.

BAR PLT=BARREN PLANTS. The percent of plants per plot that were notbarren (lack ears).

BRT STK=BRITTLE STALKS. This is a measure of the stalk breakage near thetime of pollination, and is an indication of whether a hybrid or inbredwould snap or break near the time of flowering under severe winds. Dataare presented as percentage of plants that did not snap in pairedcomparisons and on a 1 to 9 scale (9=highest resistance) inCharacteristics Charts.

BU ACR=YIELD (BUSHELS/ACRE). Yield of the grain at harvest in bushelsper acre adjusted to 15.5% moisture.

CLN=CORN LETHAL NECROSIS (synergistic interaction of maize chloroticmottle virus (MCMV) in combination with either maize dwarf mosaic virus(MDMV-A or MDMV-B) or wheat streak mosaic virus (WSMV)). A 1 to 9 visualrating indicating the resistance to Corn Lethal Necrosis. A higher scoreindicates a higher resistance.

CP=PERCENT OF CRUDE PROTEIN. The percent of dry matter that is crudeprotein in chopped whole plant forage.

COM RST=COMMON RUST (Puccinia sorghi). A 1 to 9 visual rating indicatingthe resistance to Common Rust. A higher score indicates a higherresistance.

CRM=COMPARATIVE RELATIVE MATURITY (see PRM).

CRN ERW=CORN EARWORM EAR DAMAGE SCORE. Score of ears that have been fedupon by corn earworm larvae approximately 2 weeks prior to harvest.Expressed as 1 to 9 score with 9 being no damage.

D/D=DRYDOWN. This represents the relative rate at which a hybrid willreach acceptable harvest moisture compared to other hybrids on a 1-9rating scale. A high score indicates a hybrid that dries relatively fastwhile a low score indicates a hybrid that dries slowly.

D/E or EAR RET=DROPPED EARS or EAR RETENTION SCORE. Represented in a 1to 9 scale in the Characteristics Chart, where 9 is the ratingrepresenting the least, or no, dropped ears.

DIP ERS=DIPLODIA EAR MOLD SCORES (Diplodia maydis and Diplodiamacrospora). A 1 to 9 visual rating indicating the resistance toDiplodia Ear Mold. A higher score indicates a higher resistance.

DIPROT=DIPLODIA STALK ROT SCORE. Score of stalk rot severity due toDiplodia (Diplodia maydis). Expressed as a 1 to 9 score with 9 beinghighly resistant.

DM=PERCENT OF DRY MATTER. The percent of dry material in chopped wholeplant silage.

DRP EAR=DROPPED EARS. A measure of the number of dropped ears per plotand represents the percentage of plants that did not drop ears prior toharvest.

D/T=DROUGHT TOLERANCE. This represents a 1-9 rating for droughttolerance, and is based on data obtained under stress conditions. A highscore indicates good drought tolerance and a low score indicates poordrought tolerance.

EAR HT=EAR HEIGHT. The ear height is a measure from the ground to thehighest placed developed ear node attachment and is measured in inches.This is represented in a 1 to 9 scale in the Characteristics Chart,where 9 is highest.

EAR MLD=General Ear Mold. Visual rating (1-9 score) where a “1” is verysusceptible and a “9” is very resistant. This is based on overall ratingfor ear mold of mature ears without determining the specific moldorganism, and may not be predictive for a specific ear mold.

EAR SZ=EAR SIZE. A 1 to 9 visual rating of ear size. The higher therating the larger the ear size.

EBTSTK=EARLY BRITTLE STALK. A count of the number of “snapped” plantsper plot following severe winds when the corn plant is experiencing veryrapid vegetative growth in the V5-V8 stage. Expressed as percent ofplants that did not snap.

ECB 1LF=EUROPEAN CORN BORER FIRST GENERATION LEAF FEEDING (Ostrinianubilalis). A 1 to 9 visual rating indicating the resistance topreflowering leaf feeding by first generation European Corn Borer. Ahigher score indicates a higher resistance.

ECB 2IT=EUROPEAN CORN BORER SECOND GENERATION INCHES OF TUNNELING(Ostrinia nubilias). Average inches of tunneling per plant in the stalk.

ECB 2SC=EUROPEAN CORN BORER SECOND GENERATION (Ostrinia nubilalis). A 1to 9 visual rating indicating post flowering degree of stalk breakageand other evidence of feeding by European Corn Borer, Second Generation.A higher score indicates a higher resistance.

ECB DPE=EUROPEAN CORN BORER DROPPED EARS (Ostrinia nubilalis). Droppedears due to European Corn Borer. Percentage of plants that did not dropears under second generation corn borer infestation.

E/G=EARLY GROWTH. This represents a 1 to 9 rating for early growth,scored when two leaf collars are visible.

EGRWTH=EARLY GROWTH. The relative height and size of a corn seedling atthe 2-4 leaf stage of growth. This is a visual rating (1 to 9), with 1being weak or slow growth, 5 being average growth and 9 being stronggrowth. Taller plants , wider leaves, more green mass and darker colorconstitute higher scores.

ERTLDG=EARLY ROOT LODGING. Count for severity of plants that lean from avertical axis at an approximate 30 degree angle or greater whichtypically results from strong winds prior to or around floweringrecorded within 2 weeks of a wind event. Expressed as percent of plantsnot lodged.

ERTLSC=EARLY ROOT LODGING SCORE. Score for severity of plants that leanfrom a vertical axis at an approximate 30 degree angle or greater whichtypically results from strong winds prior to or around floweringrecorded within 2 weeks of a wind event. Expressed as a 1 to 9 scorewith 9 being no lodging.

EST CNT=EARLY STAND COUNT. This is a measure of the stand establishmentin the spring and represents the number of plants that emerge on perplot basis for the inbred or hybrid.

EYE SPT=Eye Spot (Kabatiella zeae or Aureobasidium zeae). A 1 to 9visual rating indicating the resistance to Eye Spot A higher scoreindicates a higher resistance.

FALL AW=FALL ARMYWORM EAR DAMAGE SCORE. Score of ears that have been fedupon by fall armyworm larvae. Expressed as a i to 9 score with 9 beingno damage.

FUS ERS=FUSARIUM EAR ROT SCORE (Fusarium moniliforme or Fusariumsubglutinans). A 1 to 9 visual rating indicating the resistance toFusarium ear rot. A higher score indicates a higher resistance.

G/A=GRAIN APPEARANCE. Appearance of grain in the grain tank (scored downfor mold, cracks, red streak, etc.).

GDU=Growing Degree Units. Using the Barger Heat Unit Theory, thatassumes that maize growth occurs in the temperature range 50° F.-86° F.and that temperatures outside this range slow down growth; the maximumdaily heat unit accumulation is 36 and the minimum daily heat unitaccumulation is 0. The seasonal accumulation of GDU is a major factor indetermining maturity zones.

GDU PHY=GDU TO PHYSIOLOGICAL MATURITY. The number of growing degreeunits required for an inbred or hybrid line to have approximately 50percent of plants at physiological maturity from time of planting.Growing degree units are calculated by the Barger method.

GDU SHD=GDU TO SHED. The number of growing degree units (GDUs) or heatunits required for an inbred line or hybrid to have approximately 50percent of the plants shedding pollen and is measured from the time ofplanting. Growing degree units are calculated by the Barger Method,where the heat units for a 24hour period are:${GDU} = {\frac{\left( {{Max}.\quad{temp}.\quad{+ \quad{{Min}.\quad{temp}.}}} \right)}{2} - 50}$

The highest maximum temperature used is 86° F. and the lowest minimumtemperature used is 50° F. For each inbred or hybrid it takes a certainnumber of GDUs to reach various stages of plant development.

GDU SLK=GDU TO SLK. The number of growing degree units required for aninbred line or hybrid to have approximately 50 percent of the plantswith silk emergence from time of planting. Growing degree units arecalculated by the Barger Method as given in GDU SHD definition.

GIB ERS=GIBBERELLA EAR ROT (PINK MOLD) (Gibberella zaea). A 1 to 9visual rating indicating the resistance to Gibberella Ear Rot. A higherscore indicates a higher resistance.

GIBROT=GIBBERELLA STALK ROT SCORE. Score of stalk rot severity due toGibberella (Gibberella zaea). Expressed as a 1 to 9 score with 9 beinghighly resistant.

GLF SPT=Gray Leaf Spot (Cercospora zeae-maydis). A 1 to 9 visual ratingindicating the resistance to Gray Leaf Spot. A higher score indicates ahigher resistance.

GOS WLT=Goss' Wilt (Corynebacterium nebraskense). A 1 to 9 visual ratingindicating the resistance to Goss' Wilt. A higher score indicates ahigher resistance.

GRN APP=GRAIN APPEARANCE. This is a 1 to 9 rating for the generalappearance of the shelled grain as it is harvested based on such factorsas the color of harvested grain, any mold on the grain, and any crackedgrain. High scores indicate good grain quality.

H/POP=YIELD AT HIGH DENSITY. Yield ability at relatively high plantdensities on 1-9 relative rating system with a higher number indicatingthe hybrid responds well to high plant densities for yield relative toother hybrids. A 1, 5, and 9 would represent very poor, average, andvery good yield response, respectively, to increased plant density.

HC BLT=HELMINTHOSPORIUM CARBONUM LEAF BLIGHT (Helminthosporiumcarbonum). A 1 to 9 visual rating indicating the resistance toHelminthosporium infection. A higher score indicates a higherresistance.

HD SMT=Head Smut (Sphacelotheca reiliana). This score indicates thepercentage of plants not infected.

INC D/A=GROSS INCOME (DOLLARS PER ACRE). Relative income per acreassuming drying costs of two cents per point above 15.5 percent harvestmoisture and current market price per bushel.

INCOME/ACRE. Income advantage of hybrid to be patented over other hybridon per acre basis.

INC ADV=GROSS INCOME ADVANTAGE. GROSS INCOME advantage of variety #1over variety #2.

LRTLDG=LATE ROOT LODGING. Count for severity of plants that lean from avertical axis at an approximate 30 degree angle or greater whichtypically results from strong winds after flowering. Recorded prior toharvest when a root-lodging event has occurred. This lodging results inplants that are leaned or “lodged” over at the base of the plant and donot straighten or “goose-neck” back to a vertical position. Expressed aspercent of plants not lodged.

LRTLSC=LATE ROOT LODGING SCORE. Score for severity of plants that leanfrom a vertical axis at an approximate 30 degree angle or greater whichtypically results from strong winds after flowering. Recorded prior toharvest when a root-lodging event has occurred. This lodging results inplants that are leaned or “lodged” over at the base of the plant and donot straighten or “goose-neck” back to a vertical position. Expressed asa 1 to 9 score with 9 being no lodging.

L/POP=YIELD AT LOW DENSITY. Yield ability at relatively low plantdensities on a 1-9 relative system with a higher number indicating thehybrid responds well to low plant densities for yield relative to otherhybrids. A 1, 5, and 9 would represent very poor, average, and very goodyield response, respectively, to low plant density.

MDM CPX=Maize Dwarf Mosaic Complex (MDMV=Maize Dwarf Mosaic Virus andMCDV=Maize Chlorotic Dwarf Virus). A 1 to 9 visual rating indicating theresistance to Maize Dwarf Mosaic Complex. A higher score indicates ahigher resistance. MST=HARVEST MOISTURE. T he moisture is the actualpercentage moisture of the grain at harvest.

MST ADV=MOISTURE ADVANTAGE. The moisture advantage of variety #1 overvariety #2 as calculated by: MOISTURE of variety #2—MOISTURE of variety#1=MOISTURE ADVANTAGE of variety #1.

NLF BLT=Northern Leaf Blight (Helminthosporium turcicum or Exserohilumturcicum). A 1 to 9 visual rating indicating the resistance to NorthernLeaf Blight. A higher score indicates a higher resistance.

OIL=GRAIN OIL. The amount of the kernel that is oil, expressed as apercentage on a dry weight basis.

PHY CRM=CRM at physiological maturity.

PLT HT=PLANT HEIGHT. This is a measure of the height of the plant fromthe ground to the tip of the tassel in inches. This is represented as a1 to 9 scale, 9 highest, in the Characteristics Chart.

POL SC=POLLEN SCORE. A 1 to 9 visual rating indicating the amount ofpollen shed. The higher the score the more pollen shed.

POL WT=POLLEN WEIGHT. This is calculated by dry weight of tasselscollected as shedding commences minus dry weight from similar tasselsharvested after shedding is complete.

It should be understood that the inbred can, through routinemanipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplating, within thescope of the present claims.

POP K/A=PLANT POPULATIONS. Measured as 1000 s per acre.

POP ADV=PLANT POPULATION ADVANTAGE. The plant population advantage ofvariety #1 over variety #2 as calculated by PLANT POPULATION of variety#2—PLANT POPULATION of variety #1=PLANT POPULATION ADVANTAGE of variety#1.

PRM=PREDICTED Relative Maturity. This trait, predicted relativematurity, is based on the harvest moisture of the grain. The relativematurity rating is based on a known set of checks and utilizes standardlinear regression analyses and is referred to as the ComparativeRelative Maturity Rating System that is similar to the MinnesotaRelative Maturity Rating System.

PRM SHD=A relative measure of the growing degree units (GDU) required toreach 50% pollen shed. Relative values are predicted values from thelinear regression of observed GDU's on relative maturity of commercialchecks.

PRO=PROTEIN RATING. Rating on a 1 to 9 scale comparing relative amountof protein in the grain compared to hybrids of similar maturity. A “1”score difference represents a 0.4 point change in grain protein percent(e.g., 8.0% to 8.4%).

PROTEIN=GRAIN PROTEIN. The amount of the kernel that is crude protein,expressed as a percentage on a dry weight basis.

P/Y=PROTEIN/YIELD RATING. Indicates, on a 1 to 9 scale, the economicvalue of a hybrid for swine and poultry feeders. This takes into accountthe income due to yield, moisture and protein content.

ROOTS (%)=Percent of stalks NOT root lodged at harvest.

R/L or R/S=ROOT LODGING or ROOT STRENGTH SCORE. A 1 to 9 ratingindicating the level of root lodging resistance. The higher scorerepresents higher levels of resistance.

RT LDG=ROOT LODGING. Root lodging is the percentage of plants that donot root lodge: plants that lean from the vertical axis as anapproximately 30° angle or greater would be counted as root lodged.

RTL ADV=ROOT LODGING ADVANTAGE. The root lodging advantage of variety #1over variety #2.

S/L or S/S=STALK LODGING or STALK STRENGTH SCORE. A 1 to 9 ratingindicating the level of stalk lodging resistance. The higher scorerepresents higher levels of resistance.

SCT GRN SCATTER GRAIN. A 1 to 9 visual rating indicating the amount ofscatter gain (lack of pollination or kernel abortion) on the ear. Thehigher the score the less scatter grain.

SEL IND=SELECTION INDEX. The selection index gives a single measure ofthe hybrid's worth based on information for up to five traits. A maizebreeder may utilize his or her own set of traits for the selectionindex. One of the traits that is almost always included is yield. Theselection index data presented in the tables represent the mean valueaveraged across testing stations.

SIL DMP=SILAGE DRY MATTER. The percent of dry material in chopped wholeplant silage.

SLF BLT=SOUTHERN LEAF BLIGHT (Helminthosporium maydis or Bipolarismaydis). A 1 to 9 visual rating indicating the resistance to SouthernLeaf Blight. A higher score indicates a higher resistance.

SLK CRM=CRM at Silking.

SOU RST=SOUTHERN RUST (Puccinia polysora). A 1 to 9 visual ratingindicating the resistance to Southern Rust. A higher score indicates ahigher resistance.

STA GRN=STAY GREEN. Stay green is the measure of plant health near thetime of black layer formation (physiological maturity). A high scoreindicates better late-season plant health.

STAND (%)=Percent of stalks standing at harvest.

STARCH=PERCENT OF STARCH. The percent of dry matter that is starch inchopped whole plant forage.

STD ADV=STALK STANDING ADVANTAGE. The advantage of variety #1 overvariety #2 for the trait STK CNT.

STK CNT=NUMBER OF PLANTS. This is the final stand or number of plantsper plot.

STK LDG=STALK LODGING. This is the percentage of plants that did notstalk lodge (stalk breakage) as measured by either natural lodging orpushing the stalks and determining the percentage of plants that breakbelow the ear.

STKLDL=LATE SEASON STALK LODGING. A plant is considered as stalk lodgedif the stalk is broken or crimped between the ear and the ground. Thiscan be caused by any or a combination of the following: strong windslate in the season, disease pressure within the stalks, ECB damage orgenetically weak stalks. This trait should be taken when the grainmoisture content of the experiment is between 15% to 18%. Expressed aspercent of plants that did not stalk lodge.

STKLDS=REGULAR STALK LODGING SCORE. A plant is considered as stalklodged if the stalk is broken or crimped between the ear and the ground.This can be caused by any or a combination of the following: strongwinds late in the season, disease pressure within the stalks, ECB damageor genetically weak stalks. This trait should be taken just prior to orat harvest. Expressed on a 1 to 9 scale with 9 being no lodging.

STR RWH=PERCENT OF STARCH. This is the percent of dry matter that isstarch in chopped whole plant forage as predicted by Near InfraredSpectroscopy.

STW WLT=Stewart's Wilt (Erwinia stewartii). A 1 to 9 visual ratingindicating the resistance to Stewart's Wilt. A higher score indicates ahigher resistance.

SW C/B=SOUTHWESTERN CORN BORER DAMAGE SCORE. Score of plants that havebeen girdled (hollowed out) at the base by SWCB feeding. The score isbased on the count of plants that break as measured against the STKCNTjust prior to harvest. Expressed as 1 to 9 score with 9 being no damage.

TAS BLS=TASSEL BLAST. A 1 to 9 visual rating was used to measure thedegree of blasting (necrosis due to heat stress) of the tassel at thetime of flowering. A 1 would indicate a very high level of blasting attime of flowering, while a 9 would have no tassel blasting.

TAS SZ=TASSEL SIZE. A 1 to 9 visual rating was used to indicate therelative size of the tassel. The higher the rating the larger thetassel.

TAS WT=TASSEL WEIGHT. This is the average weight of a tassel (grams)just prior to pollen shed.

TDM/HA=TOTAL DRY MATTER PER HECTARE. Yield of total dry plant materialin metric to ns per hectare.

TEX EAR=EAR TEXTURE. A 1 to 9 visual rating was used to indicate therelative hardness (smoothness of crown) of mature grain. A 1 would bevery soft (extreme dent) while a 9 would be very hard (flinty or verysmooth crown).

TIL LER=TILLERS. A count of the number of tillers per plot that couldpossibly shed pollen was taken. Data are given as a percentage oftillers: number of tillers per plot divided by number of plants perplot.

TST WT (CHARACTERISTICS CHART)=Test weight on a 1 to 9 rating scale witha 9 being the highest rating.

TST WT=TEST WEIGHT (UNADJUSTED). The measure of the weight of the grainin pounds for a given volume (bushel).

TST WTA=TEST WEIGHT ADJUSTED. The measure of the weight of the grain inpounds for a given volume (bushel) adjusted for 15.5 percent moisture.

TSW ADV=TEST WEIGHT ADVANTAGE. The test weight advantage of variety #1over variety #2.

WIN M % PERCENT MOISTURE WINS.

WIN Y %=PERCENT YIELD WINS.

YIELD=YIELD OF SILAGE. Yield in tons per acre at 30% dry matter.

YLD=YIELD. It is the same as BU ACR ABS.

YLD ADV=YIELD ADVANTAGE. The yield advantage of variety #1 over variety#2 as calculated by: YIELD of variety #1—YIELD variety #2=yieldadvantage of variety #1.

YLD SC=YIELD SCORE. A 1 to 9 visual rating was used to give a relativerating for yield based on plot ear piles. The higher the rating thegreater visual yield appearance.

DETAILED DESCRIPTION OF THE INVENTION

Pioneer Brand Hybrid X1069G has excellent yield potential. The hybridshows good stalk and root lodging resistance. Hybrid X1069G alsoexhibits good early growth, stay green and test weight. X1069G furtherdemonstrates very good dry down, ear retention and husk cover, anddependable drought stress tolerance. Hybrid X1069G demonstrates a gooddisease resistance package with moderate resistance to Gray Leaf Spot,Northern Leaf Blight, Eye Spot, Fusarium Ear Rot, Gibberella Ear Rot,and Common Rust, excellent resistance to head smut, and moderateresistance to European Corn Borer first and second generation. It isparticularly suited to the Central Corn Belt, Northwest, Northcentral,Northeastern, and Western regions of the United States.

Pioneer Brand Hybrid X1069G is a single cross, yellow endosperm, dentmaize hybrid. Hybrid X1069G has a relative maturity of approximately 105(106 for physiological maturity) based on the Comparative RelativeMaturity Rating System for harvest moisture of grain.

This hybrid has the following characteristics based on the datacollected primarily at Johnston, Iowa.

TABLE 1 VARIETY DESCRIPTION INFORMATION VARIETY = X1069G 1. TYPE:(describe intermediate types in Commerts section): 2 1 = Sweet 2 = Dent3 = Flint 4 = Flour 5 = Pop 6 = Ornamental 2. MATURITY: DAYS HEAT UNITS065 1,225.0 From emergence to 50% of plants in silk 069 1,294.0 Fromemergence to 50% of plants in pollen 005 0,113.3 From 10% to 90% pollenshed From 50% silk to harvest at 25% moisture 3. PLANT: Standard SampleDeviation Size 0,274.2 cm Plant Height (to tassel tip) 3.51 15 0,109.3cm Ear Height (to base of top ear node) 6.66 15 0,019.3 cm Length of TopEar Internode 1.29 15 0.0 Average Number of Tillers 0.00 3 1.0 AverageNumber of Ears per Stalk 0.07 3 2.0 Anthocyanin of Brace Roots: 1 =Absent 2 = Faint 3 = Moderate 4 = Dark 5 = Very Dark 4. LEAF: StandardSample Deviation Size 011.3 cm Width of Ear Node Leaf 0.99 15 098.3 cmLength of Ear Node Leaf 1.81 15 06.5 Number of leaves above top ear 0.4215 018.5 Degrees Leaf Angle (measure from 2nd leaf above 5.00 15 ear atanthesis to stalk above leaf) 03 Leaf Color Dark Green (Munsell code)7.5GY34 1.3 Leaf Sheath Pubescence (Rate on scale from 1 = none to 9 =like peach fuzz) Marginal Waves (Rate on scale from 1 = none to 9 =many) Longitudinal Creases (Rate on scale from 1 = none to 9 = many) 5.TASSEL: Standard Sample Deviation Size 04.1 Number of Primary LateralBranches 1.17 15 019.6 Branch Angle from Central Spike 3.12 15 62.3 cmTassel Length (from top leaf collar to tassel tip) 3.00 15 4.7 PollenShed (rate on scale from 0 = male sterile to 9 = heavy shed) 07 AntherColor Yellow (Munsell code) 7.5Y8.56 01 Glume Color Light Green (Munsellcode) 5GY58 1.0 Bar Glumes (Glume Bands); 1 = Absent 2 = Present 20 cmPeduncle Length (cm. from top leaf to basal branches) 6a. EAR (UnhuskedData): 1 Silk color (3 days after emergence) Light Green (Munsell code)2.5GY89 3 Fresh Husk Color (25 days after 50% Silking) Dark Green(Munsell code) 5GY56 21 Dry Husk Color (65 days after 50% silking) Buff(Munsell code) 2.5Y92 3 Position of Ear at Dry Husk Stage: 1 = Upright 2= Horizontal 3 = Pendant Pendant 5 Husk Tightness (Rate of Scale from 1= very loose to 9 = very tight) 2 Husk Extension (at harvest): 1 = Short(ears exposed) 2 = Medium (<8 cm) 3 = Long (8-10 cm beyond ear tip) 4 =Very Long (>10 cm) Medium 6b. EAR (Husked Ear Data): Standard SampleDeviation Size 17 cm Ear Length 0.58 15 47 mm Ear Diameter at mid-point1.00 15 191 gm Ear Weight 24.19 15 17 Number of Kernel Rows 0.58 15 2Kernel Rows: 1 = Indistinct 2 = Distinct Distinct 2 Row Alignment: 1 =Straight 2 = Slightly Curved 3 = Spiral Slightly Curved 11 cm ShankLength 1.00 15 2 Ear Taper: 1 = Slight 2 = Average 3 = Extreme Average7. KERNEL (Dried): Standard Sample Deviation Size 12 mm Kernel Length1.00 15 8 mm Kernel Width 0.00 15 4 mm Kernel Thickness 0.58 15 45 %Round Kernels (Shape Grade) 2.00 3 1 Aleurone Color Pattern: 1 =Homozygous 2 = Segregating Homozygous 7 Aluerone Color Yellow (Munsellcode) 1.25Y714 7 Hard Endosperm Color Yellow (Munsell code) 1.25Y714 3Endosperm Type: Normal Starch 1 = Sweet (Su1) 2 = Extra Sweet (sh2) 3 =Normal Starch 4 = High Amylose Starch 5 = Waxy Starch 6 = High Protein 7= High Lysine 8 = Super Sweet (se) 9 = High Oil 10 = Other     30 gmWeight per 100 Kernels (unsized sample) 4.00 3 8. COB: Standard SampleDeviation Size 26 mm Cob Diameter at mid-point 0.58 15 14 Cob Color Red(Munsell code) 10R56 9. DISEASE RESISTANCE (Rate from 1 (mostsusceptible) to 9 (most resistant); leave blank     if not tested; leaveRace or Strain Options blank if polygenic): A. Leaf Blights, Wilts, andLocal Infection Diseases Anthracnose Leaf Blight (Colletotrichumgraminicola) 5 Common Rust (Puccinia sorghi) Common Smut (Ustilagomaydis) 5 Eyespot (Kabatiella zeae) Goss's Wilt (Clavibactermichiganense spp. nebraskense) 5 Gray Leaf Spot (Cercospora zeae-maydis)Helminthosporium Leaf Spot (Bipolaris zeicola) Race     5 Northern LeafBlight (Exserohilum turcicum) Race     Southern Leaf Blight (Bipolarismaydis) Race     Southern Rust (Puccinia polysora) 4 Stewart's Wilt(Erwinia stewartii) Other (Specify)     B. Systemic Diseases Corn LethalNecrosis (MCMV and MDMV) Head Smut (Sphacelotheca reiliana) MaizeChlorotic Dwarf Virus (MDV) Maize Chlorotic Mottle Virus (MCMV) MaizeDwarf Mosaic Virus (MDMV) Sorghum Downy Mildew of Corn(Peronosclerospora sorghi) Other (Specify)     C. Stalk Rots 5Anthracnose Stalk Rot (Colletotrichum graminicola) Diplodia Stalk Rot(Stenocarpella maydis) Fusarium Stalk Rot (Fusarium moniliforme)Gibberella Stalk Rot (Gibberella zeae) Other (Specify)     D. Ear andKernel Rots Aspergillus Ear and Kernel Rot (Aspergillus flavus) 2Diplodia Ear Rot (Stenocarpella maydis) 5 Fusarium Ear and Kernel Rot(Fusarium moniliforme) 6 Gibberella Ear Rot (Gibberella zeae) Other(Specify)     10. INSECT RESISTANCE (Rate from 1 (most susceptible) to 9(most resistant);     (leave blank if not tested): Banks grass Mite(Oligonychus pratensis) Corn Worm (Helicoverpa zea) Leaf Feeding SilkFeeding mg larval wt. Ear Damage Corn Leaf Aphid (Rhopalosiphum maidis)Corn Sap Beetle (Carpophilus dimidiatus) European Corn Borer (Ostrinianubilalis) 6 1st Generaton (Typically Whorl Leaf Feeding) 5 2ndGeneration (Typically Leaf Sheath-Collar Feeding) Stalk Tunneling cmtunneled/plant Fall Armyworm (Spodoptera fruqiperda) Leaf Feeding SilkFeeding mg larval wt. Maize Weevil (Sitophilus zeamaize) NorthernRootworm (Diabrotica barberi) Southern Rootworm (Diabroticaundecimpunctata) Southwestern Corn Borer (Diatreaea grandiosella) LeafFeeding Stalk Tunneling cm tunneled/plant Two-spotted Spider Mite(Tetranychus urticae) Western Rootworm (Diabrotica virgifrea virgifera)Other (Specify)     11. AGRONOMIC TRAITS: 4 Staygreen (at 65 days afteranthesis) (Rate on a scale from 1 = worst to 9 = excellent) 0.1 %Dropped Ears (at 66 days after anthesis) % Pre-anthesis Brittle Snapping% Pre-anthesis Root Lodging 3.2 Post-anthesis Root Lodging (at 65 daysafter anthesis) 10,731 Kg/ha Yield (at 12-13% grain moisture) *Ininterpreting the foregoing color designations, reference may be made tothe Munsell Glossy Book of Color, a standard color reference.

Research Comparisons for Pioneer Hybrid X1069G

Comparisons of characteristics for Pioneer Brand Hybrid X1069G were madeagainst Pioneer Brand Hybrids 3563, 36B08, 35R57, 35P12, 34G81, and34G13.

Table 2A compares Pioneer Brand Hybrid X1069G and Pioneer Brand Hybrid3563, a hybrid of similar maturity. The table shows that hybrid X1069Gis significantly higher yielding than hybrid 3563. Hybrid X1069G isearlier to mature with a significantly lower number of growing degreeunits to pollen shed and to silk and a significantly later predictedrelative maturity score than hybrid 3563. Hybrid X1069G also exhibitssignificantly shorter plant stature with significantly superiortolerance to stalk lodging (STK LDS ABS) than hybrid 3563. Hybrid X1069Gexhibits significantly superior tolerance to Gray Leaf Spot, andsignificantly better resistance Anthracnose Stalk Rot than hybrid 3563.

Table 2B compares Pioneer Brand Hybrid X1069G and Pioneer Brand Hybrid36B08, a closely related hybrid of similar maturity. The table indicatesthat hybrid X1069G is significantly higher yielding with significantlylower harvest moisture than hybrid 36B08. Hybrid X1069G is later tomature with a significantly higher number of growing degree units topollen shed and to silk than hybrid 36B08. Hybrid X1069G also exhibitssignificantly taller plant stature than hybrid 36B08.

Table 2C compares Pioneer Brand Hybrid X1069G and Pioneer Brand Hybrid35R57, a hybrid of similar maturity. The results indicate that hybridX1069G is significantly higher yielding than hybrid 35R57. Hybrid X1069Gexhibits a significantly lower number of growing degree units to silkand significantly superior tolerance to Gray Leaf Spot than hybrid35R57.

Table 2D compares Pioneer Brand Hybrid X1069G and Pioneer Brand Hybrid35P12, a hybrid of similar maturity. According to the results hybridX1069G is similar in yield but exhibits significantly lower harvestmoisture and a significantly higher test weight (TST WT ABS and TST WTAABS) than hybrid 35P12. Hybrid X1069G exhibits a significantly highernumber of growing degree units to pollen shed and significantly lowerear placement than hybrid 35P12. Hybrid X1069G also exhibitssignificantly superior tolerance to late season stalk lodging andsignificantly superior tolerance to Gray Leaf Spot than hybrid 35P12.

Table 2E compares Pioneer Brand Hybrid X1069G and Pioneer Brand Hybrid34G81, a hybrid of similar maturity. That table shows that hybrid XI069G is significantly higher yielding with significantly lower harvestmoisture and a significantly higher test weight (TST WT ABS and TST WTAABS) than hybrid 34G81. Hybrid X1069G is earlier to mature with asignificantly lower number of growing degree units to pollen shed and tosilk than hybrid 34G81. Hybrid X1069G also exhibits significantlyshorter plant stature with significantly lower ear placement as well assignificantly superior tolerance to root lodging than hybrid 34G81.Hybrid X1069G demonstrates significantly superior early growth andsignificantly superior tolerance to Fusarium Ear Rot as well as asignificantly larger dusk cover than hybrid 34G81.

Table 2F compares Pioneer Brand Hybrid X1069G and Pioneer Brand Hybrid34G13, a closely related hybrid of similar maturity. The results showthat hybrid X1069G is similar in yield but exhibits significantly lowerharvest moisture than hybrid 34G13. Hybrid X1069G also exhibitssignificantly lower number of growing degree units to pollen shed andsignificantly earlier predicted relative maturity score than hybrid34G13. Hybrid X1069G also exhibits significantly taller plant staturethan hybrid 34G13.

TABLE 2A HYBRID COMPARISON REPORT VARIETY #1 = X10E9G VARIETY #2 = 3563PRM BU BU TST GDU GDU PLT PRM SHD ACR ACR MST WTA SHD SLK HT ABS ABS ABS% MN % MN ABS % MN % MN % MN TOTAL SUM 1 105 105 183.5 105 96 56.1 99 9897 2 103 106 163.2 94 95 56.8 101 101 104 LOCS 5 3 178 178 182 118 38 3153 REPS 5 3 185 185 169 123 46 39 60 DIFF 2 2 20.3 12 4 0.7 2 4 7 PR > T.004# .055* .000# .000# .000# .000# .000# .000# .000# EAR ERT RT LRT STKSTK STK BRT ABT HT LSC LDG LSC LDS LDG LDL STK STK % MN ABS % MN ABS ABS% MN % MN % MN % MN TOTAL SUM 1 98 3.0 105 7.8 7.2 99 104 102 94 2 997.0 105 7.4 6.3 100 92 97 89 LOCS 53 1 14 12 59 66 29 14 9 REPS 60 1 1513 65 66 46 14 33 DIFF 1 4.0 0 0.4 0.8 1 11 5 5 PR > T .661 .999 .422.017+ .777 .112 .234 .612 EGR STA DRP TST STK EST GLF NLF STW WTH GRNEAR WT CMT CNT SPT BLT WLT % MN % MN % MN ABS % MN % MN ABS ABS ABSTOTAL SUM 1 99 87 100 56.2 99 102 4.8 4.9 4.0 2 103 91 100 57.1 101 1023.6 5.1 4.0 LOCS 33 57 15 117 259 6 14 8 2 REPS 34 61 16 122 339 6 20 124 DIFF 3 3 0 0.9 2 0 1.1 0.1 0.0 PR > T .395 .530 .999 .000# .000# .999.038+ .896 .999 ANT HD FUS GIB DIP EYE ECB ECB HSK GIB ROT SMT ERS ERSERS SPT 1LF 2SC CVR ROT ABS ABS ABS ABS ABS ABS ABS ABS ABS ABS TOTALSUM 1 4.6 99.2 4.6 6.5 2.3 5.0 5.5 4.8 6.4 7.0 2 3.5 97.8 4.5 4.8 3.35.0 4.0 6.3 6.3 6.0 LOCS 10 3 4 3 3 1 2 4 14 1 REPS 16 6 5 4 6 2 3 6 172 DIFF 1.1 1.4 0.1 1.7 1.0 0.0 1.5 1.5 0.1 1.0 PR > T .030+ .394 .824.199 .438 .205 .319 .702 *= 10% SIG += 5% SIG #= 1% SIG

TABLE 2B HYBRID COMPARISON REPORT VARIETY #1 = X1069G VARIETY #2 = 36B08PRM BU BU TST GDU GDU PLT PRM SHD ACR ACR MST WTA SHD SLK HT ABS ABS ABS% MN % MN ABS % MN % MN % MN TOTAL SUM 1 104 104 167.9 105 97 54.9 99 9897 2 104 103 161.6 101 99 55.0 97 96 92 LOCS 7 1 110 110 113 87 19 16 40REPS 7 1 125 125 129 91 25 22 47 DIFF 0 1 6.3 4 2 0.1 2 1 4 PR > T .999.000# .000# .001# .399 .000# .004# .000# EAR RT LRT STK STK STK EBT BRTABT HT LDG LSC LDS LDG LDL STK STK STK % MN % MN ABS ABS % MN % MN % MN% MN % MN TOTAL SUM 1 99 110 8.4 7.2 97 103 100 101 86 2 96 145 7.6 7.399 111 101 101 97 LOCS 31 2 5 43 11 14 8 2 4 REPS 38 3 5 48 13 23 32 222 DIFF 3 35 0.8 0.1 2 8 2 0 9 PR > T .280 .090* .374 .670 .722 .375.883 .999 .190 EGR STA DRP STK EST GLF NLF ANT HD WTH GRN EAR CNT CNTSPT BLT ROT SMT % MN % MN % MN % MN % MN ABS ABS ABS ABS TOTAL SUM 1 9183 100 100 96 4.1 5.3 4.8 99.8 2 110 114 100 101 105 4.4 7.4 4.7 99.6LOCS 18 38 2 158 1 5 6 7 7 REPS 25 41 2 242 1 9 10 9 13 DIFF 19 31 0 1 80.3 2.1 0.1 0.2 PR > T .003# .000# .999 .050* .374 .035+ .930 .627 FUSGIB DIP ECB ECB HSK GIB ERS ERS ERS 1LF 2SC CVR ROT ABS ABS ABS ABS ABSABS ABS TOTAL SUM 1 4.5 5.8 2.3 5.0 4.8 6.3 7.0 2 3.7 6.8 2.0 6.0 5.15.9 8.5 LOCS 3 2 2 1 4 8 1 REPS 5 3 4 2 6 11 2 DIFF 0.8 1.0 0.3 1.0 0.40.4 1.5 PR > T .423 .000# .874 .547 .602 *= 10% SIG += 5% SIG #= 1% SIG

TABLE 2C HYBRID COMPARISON REPORT VARIETY #1 = X1069G VARIETY #2 = 35R57PRM BU BU TST GDU GDU PLT PRM SHD ACR ACR MST WTA SHD SLK HT ABS ABS ABS% MN % MN ABS % MN % MN % MN TOTAL SUM 1 106 105 184.4 105 98 56.0 99 9897 2 105 105 172.2 98 98 55.8 100 100 98 LOCS 5 3 178 178 184 117 37 3053 REPS 5 3 182 182 188 117 45 38 59 DIFF 0 1 12.1 7 0 0.2 0 2 1 PR > T.999 .243 .000# .000# .999 .157 .999 .000# .083* EAR ERT RT LRT STK STKSTK BRT ABT HT LSC LDS LSG LDS LDG LDL STK STK % MN ABS % MN ABS ABS %MN % MN % MN % MN TOTAL SUM 1 98 3.0 105 7.9 7.2 99 104 102 94 2 96 3.0104 7.0 7.2 104 105 105 107 LOCS 52 1 14 13 55 65 29 14 9 REPS 58 1 1514 55 65 46 14 35 DIPF 2 0.0 1 0.9 0.1 6 2 3 13 PR > T .149 .764 .224.814 .035+ .802 .417 .153 EGR STA DRP TST STK EST GLF NLF STW WTH GRNEAR WT CNT CNT SPT BLT WLT % MN % MN % MN ABS % MN % MN ABS ABS ABSTOTAL SUM 1 99 87 100 56.1 99 102 4.8 4.9 4.0 2 97 82 99 56.0 101 1003.7 6.0 6.3 LOCS 32 53 15 117 257 6 14 8 2 REPS 32 54 16 117 337 6 20 124 DIFF 2 5 1 0.2 2 2 1.1 1.1 2.3 PR > T .638 .409 .123 .265 .000# .450.015+ .139 .323 ANT HD FUS GIB DIP EYE ECB ECB HSK GIB ROT SMT ERS ERSERS SPT 1LF 2SC CVR ROT ABS ABS ABS ABS ABS ABS ABS ABS ABS ABS TOTALSUM 1 4.7 99.2 4.8 5.9 2.3 5.0 5.5 4.8 6.4 7.0 2 4.7 91.3 4.1 4.3 4.85.5 5.0 4.1 6.0 5.5 LOCS 10 3 4 4 3 1 2 4 14 1 REPS 15 6 5 5 6 2 3 6 172 DIFF 0.0 7.9 0.6 1.6 2.5 0.5 0.5 0.6 0.4 1.5 PR > T .999 .213 .391.135 .013+ .500 .278 .171 *= 10% SIG += 5% SIG #= 1% SIG

TABLE 2D HYBRID COMPARISON REPORT VARIETY #1 = X1069G VARIETY #2 = 35P12PRM BU BU TST GDU GDU PLT PRM SHD ACR ACR MST WTA SHD SLK HT ABS ABS ABS% MN % MN ABS % MN % MN % MN TOTAL SUM 1 105 105 172.0 104 97 55.9 98 9896 2 105 104 169.7 103 101 54.6 97 98 97 LOCS 21 10 305 305 311 226 6135 98 REPS 21 10 341 341 348 249 78 43 116 DIFF 1 1 2.2 1 3 1.3 1 0 1PR > T .068* .107 .079* .236 .000# .000# .000# .999 .050* EAR ERT RT LRTSTK STK STK EBT BRT HT LSC LDG LSC LDS LDG LDL STK STK % MN ABS % MN ABSABS % MN % MN % MN % MN TOTAL SUM 1 97 4.4 104 8.2 7.2 100 104 100 96 299 5.2 113 7.6 6.9 97 86 97 102 LOCS 84 5 7 20 129 77 48 8 10 REPS 102 58 21 155 80 65 32 10 DIFF 2 0.8 9 0.6 0.3 3 17 2 5 PR > T .033+ .178.189 .142 .116 .149 .028+ .644 .157 ABT EGR STA DRP TST STK EST GLF NLFSTK WTH GRN EAR WT CNT CNT SPT BLT % MN % MN % MN % MN ABS % MN % MN ABSABS TOTAL SUM 1 94 100 82 100 55.9 100 101 4.7 4.9 2 115 102 119 10054.7 102 104 3.3 6.3 LOCS 7 40 87 15 226 447 3 15 8 REPS 31 43 100 16249 602 3 21 12 DIFF 22 2 37 0 1.2 2 3 1.4 1.4 PR > T .092* .590 .000#.999 .000# .000# .088# .005# .095# STW ANT HD FUS GIB DIP EYE ECB ECBHSK GIB WLT ROT SMT ERS ERS ERS SPT 1LF 2SC CVR ROT ABS ABS ABS ABS ABSABS ABS ABS ABS ABS ABS TOTAL SUM 1 4.0 4.6 99.2 4.8 5.9 2.3 5.0 5.5 4.86.3 7.0 2 5.5 4.7 97.2 4.8 6.0 4.0 5.0 6.8 4.1 6.8 7.5 LOCS 2 10 3 4 4 31 2 4 13 1 REPS 4 16 6 7 5 6 2 3 6 16 2 DIFF 1.5 0.1 2.0 0.0 0.1 1.7 0.01.3 0.6 0.6 0.5 PR > T .374 .877 .423 .999 .789 .199 .344 .278 .219 *=10% SIG += 5% SIG #=1% SIG

TABLE 2E HYBRID COMPARISON REPORT VARIETY #1 = X1069G VARIETY #2 = 34G81PRM BU BU TST GDU GDU PLT PRM SHD ACR ACR MST WTA SHD SLK HT ABS ABS ABS% MN % MN ABS % MN % MN % MN TOTAL SUM 1 105 105 172.9 101 97 55.9 98 9896 2 105 105 167.2 102 99 55.6 99 100 99 LOCS 22 10 338 338 343 232 6736 107 REPS 22 10 370 370 376 255 84 44 125 DIFF 0 1 5.7 3 2 0.3 1 2 2PR > T .999 .164 .000# .000# 000# .000# .001# .000# .000# EAR ERT RT LRTSTK STK STK BRT ABT HT LSC LDS LSC LDS LDG LDL STK STK % MN ABS % MN ABSABS % MN % MN % MN % MN TOTAL SUM 1 97 4.4 103 8.2 7.2 100 104 101 96 2100 4.2 94 7.3 7.1 101 106 102 110 LOCS 93 5 27 20 130 96 51 20 10 REPS111 5 28 21 156 99 68 20 37 DIFF 3 0.2 9 0.8 0.1 1 2 1 14 PR > T .023+.778 .036+ .136 .568 .715 .735 .625 .042+ EGR STA DRP TST STK EST GLFNLF STW WTH GRN EAR WT CNT CNT SPT BLT WLT % MN % MN % MN ABS % MN % MNABS ABS ABS TOTAL SUM 1 101 82 100 56.0 100 103 4.7 4.9 4.0 2 86 110 10055.6 101 100 4.6 7.2 6.3 LOCS 42 92 15 232 481 8 15 8 2 REPS 45 105 16255 613 8 21 12 4 DIFF 15 27 1 0.3 1 3 0.1 2.3 2.3 PR > T .004# .000#.083* .000# .000# .179 .535 .002# .323 ANT HD FUS GIB DIP EYE COM ECBECB HSK GIB ROT SMT ERS ERS ERS SPT RST 1LF 2SC CVR ROT ABS ABS ABS ABSABS ABS ABS ABS ABS ABS ABS TOTAL SUM 1 4.7 99.2 4.8 5.9 2.3 5.0 4.5 5.54.8 6.4 7.0 2 4.6 99.5 3.3 4.9 4.2 6.5 5.3 5.0 4.9 5.4 7.5 LOCS 10 3 4 43 1 4 2 4 14 1 REPS 15 6 7 5 6 2 4 3 6 17 2 DIFF 0.1 0.4 1.5 1.0 1.8 1.50.8 0.5 0.1 1.0 0.5 PR > T .937 .767 .005# .092* .053* .058* .500 .718.002# *= 10% SIG += 5% SIG #= 1% SIG

TABLE 2F HYBRID COMPARISON REPORT VARIETY #1 = X1069G VARIETY #2 = 34G13PRM BU BU TST GDU GDU PLT PRM SHD ACR ACR MST WTA SHD SLK HT ABS ABS ABS% MN % MN ABS % MN % MN % MN TOTAL SUM 1 105 105 179.9 104 98 56.1 99 9897 2 108 105 182.0 106 108 56.1 99 99 95 LOCS 8 5 183 183 189 128 46 3359 REPS 8 5 210 210 217 145 60 41 74 DIFF 4 0 2.1 2 10 0.0 0 2 2 PR > T.001# .999 .164 .080* .000# .999 .999 .000# .006# EAR ERT RT LRT STK STKSTK EBT BRT HT LSC LDG LSC LDS LDG LDL STK STK % MN ABS % MN ABS ABS %MN % MN % MN % MN TOTAL SUM 1 98 3.0 104 7.9 7.2 99 103 100 95 2 98 3.099 7.8 8.1 107 119 111 103 LOCS 59 1 7 13 75 62 37 8 5 REPS 74 1 8 14 9664 54 32 5 DIFF 0 0.0 5 0.1 0.9 8 15 11 7 PR > T .999 .570 .886 .000#.006# .020+ .570 .278 ABT EGR STA DRP TST STK EST GLF NLF STK WTH GRNEAR WT CNT CNT SPT BLT % MN % MN % MN % MN ABS % MN % MN ABS ABS TOTALSUM 1 89 98 80 100 56.2 98 100 4.8 4.9 2 97 102 129 100 55.9 101 95 4.54.7 LOCS 6 34 63 15 128 289 2 14 8 REPS 30 37 73 16 145 432 2 20 12 DIFF8 4 49 0 0.3 2 5 0.3 0.3 PR > T .358 .393 .000# .999 .064* .000# .215.283 .695 STW ANT HD FUS GIB DIP EYE ECB ECB HSK GIB WLT ROT SMT ERS ERSRRS SPT 1LP 2SC CVR ROT ABS ABS ABS ABS ABS ABS ABS ABS ABS ABS ABSTOTAL SUM 1 4.0 4.6 99.2 4.8 5.9 2.3 5.0 5.5 4.8 6.3 7.0 2 5.5 6.2 94.93.9 6.4 1.8 6.5 6.8 4.3 5.3 6.5 LOCS 2 10 3 4 4 3 1 2 4 13 1 REPS 4 16 67 5 6 2 3 6 16 2 DIFF 1.5 1.6 4.3 0.9 0.5 0.5 1.5 1.3 0.5 1.0 0.5 PR> T.374 .009# .055# .310 .182 .423 .126 .423 .017+ *= 10% SIG += 5% SIG #=1% SIG

Strip Test Data for Hybrid X1069G

Comparison data was collected from strip tests that were grown byfarmers. Each hybrid was grown in strips of 4, 6, 8, 12, etc. rows infields depending on the size of the planter used. The data was collectedfrom strip tests that had the hybrids in the same area and weighed. Themoisture percentage was determined and bushels per acre was adjusted to15.5 percent moisture. The number of comparisons represent the number oflocations or replications for the two hybrids that were grown in thesame field in close porximity and compared.

Comparison strip testing was done between Pioneer Brand Hybrid X1069Gand Pioneer Brand Hybrids 34G13, 34G81, 35P12, 35R57, 3563, and 36B08.The comparisons come from all the hybrid's adapted growing areas in theUnited States.

These results are presented in Table 3. As can be seen from the tablehybrid X1069G demonstrates an average yield and income advantage overthe comparison hybrids. The average yield advantage was 1.2 bushels peracre and the average income advantage was $4.12 per acre. HybridX1069G's advantage for these and other characteristics over thesehybrids will make it an important addition for most of the areas wherethese hybrids are grown.

TABLE 3 2000 PERFORMANCE COMPARISON REPORT FOR CORN 1 YEAR SUMMARY OFALL STANDARD TEST TYPES Pop Income/ K/ Stand Roots Test Brand ProductYield Moist Acre Acre (%) (%) Wt Pioneer X1069G 171.6 16.8 334.77 26.978 97 57.6 Pioneer 34G13 174.4 18.8 334.34 27.9 87 98 57.7 Advantage−2.8 2.0 .43 −1.0 −9 −1 −.1 Number of 104 104 104 92 73 54 98Comparisons Percent Wins 43 89 46 18 21 7 36 Probability of 97 99 14 9999 85 66 Difference Pioneer X1069G 167.7 17.5 325.15 26.9 80 97 57.3Pioneer 34G81 169.0 18.1 326.37 27.5 83 90 57.3 Advantage −1.3 .6 −1.22−.6 −3 7 .0 Number of 149 149 149 129 85 65 141 Comparisons Percent Wins46 68 50 29 31 37 39 Probability of 84 99 48 99 65 88 48 DifferencePioneer X1069G 166.2 17.5 322.41 26.8 79 97 57.3 Pioneer 35P12 166.418.3 321.11 27.1 78 95 56.5 Advantage −.2 .8 1.30 −.3 1 2 .8 Number of150 150 150 130 85 65 142 Comparisons Percent Wins 55 68 59 34 42 20 64Probability of 13 99 46 96 61 34 99 Difference Pioneer X1069G 189.7 17.9366.82 28.4 89 99 57.3 Pioneer 35R57 184.1 17.0 358.21 29.3 89 99 57.2Advantage 5.6 −.9 8.61 −.9 0 0 .1 Number of 25 25 25 23 17 15 25Comparisons Percent Wins 76 24 64 30 29 7 44 Probability of 96 99 89 3258 66 71 Difference Pioneer X1069G 162.6 16.3 318.74 26.4 73 96 57.7Pioneer 3563 148.0 15.6 291.61 27.1 70 96 58.4 Advantage 14.6 −.7 27.13−.7 3 0 −.7 Number of 73 73 73 64 50 34 67 Comparisons Percent Wins 8926 89 23 58 9 15 Probability of 99 99 99 99 54 78 99 Difference PioneerX1069G 146.9 20.0 278.30 24.9 96 100 57.6 Pioneer 36B08 151.3 19.3288.77 26.3 98 100 58.3 Advantage −4.4 −.7 −10.47 −1.4 −2 0 −.7 Numberof 13 13 13 8 5 3 12 Comparisons Percent Wins 38 31 38 25 20 0 33Probability of 65 90 77 46 41 − 95 Difference Pioneer X1069G 167.9 17.3326.23 26.9 79 97 57.4 Weighted Avg 166.7 17.9 322.11 27.5 81 95 57.3Advantage 1.2 .6 4.12 −.6 −2 2 .1 Number of 514 514 514 446 315 236 485Comparisons Percent Wins 55 63 58 28 36 19 42 Probability of 95 99 99 9943 42 98 Difference NOTE: The probability values are useful in analyzingif there is a “real” difference in the genetic potential of the productsinvolved. High values are desirable, with 95% considered significant forreal differences.

Comparison of Key Characteristics for Hybrid X1069G

Characteristics of Pioneer Hybrid X1069G are compared to Pioneer Hybrids3563, 36B08, 35R57, 35P12, 34G81, and 34G13 in Table 4. The values givenfor most traits are on a 1-9 basis. In these cases 9 would beoutstanding, while 1 would be poor for the given characteristics. Table4 shows that hybrid X1069G exhibits a unique combination of outstandingyield, outstanding head smut tolerance and excellent dry down. HybridX1069G's favorable agronomic characteristics should make it an importanthybrid to its area of adaptation.

TABLE 4 Hybrid Patent Comparisons−Characteristics Pioneer Hybrid X1069Gvs. Pioneer Hybrids 3563, 36B08, 35R57, 35P12, 34G81, 34G13 SILK PHY GDUGDU VARIETY CRM CRM CRM SILK PHY YLD H/POP L/POP D/D S/S X1069G 105 105106 1310 2550 9 8 6 3563 103 105 105 1310 2530 7 7 8 7 4 36B08 103 100102 1250 2450 9 9 8 5 6 35R57 104 104 105 1300 2530 9 9 8 7 6 35P12 104103 105 1280 2530 9 9 8 6 4 34G81 106 106 106 1320 2550 8 8 8 7 6 34G13109 105 107 1310 2580 9 9 8 7 7 HSK STA TST PLT EAR EAR BRT VARIETY CVRR/S GRN D/T WT E/G HT HT RET STK X1069G 6 6 6 7 6 6 6 5 6 3563 7 7 6 7 84 7 4 7 4 36B08 7 7 7 8 7 7 3 3 5 6 35R57 6 5 5 8 6 5 5 4 4 4 35P12 8 67 8 4 7 5 5 6 6 34G81 4 3 6 8 6 5 6 5 5 5 34G13 5 6 7 8 6 5 5 5 6 GLFNLF SLF GOS STW ANT HD FUS VARIETY SPT BLT BLT WLT WLT ROT SMT CLN MDMERS X1069G 5 5 4 4 9 5 3563 3 5 3 8 4 4 8 3 3 6 36B08 3 5 7 4 4 7 6 435R57 3 6 7 6 4 5 3 5 35P12 3 6 7 5 5 8 4 4 34G81 4 7 5 8 6 5 9 2 3 434G13 4 4 7 6 6 7 4 4 GIB DIP EYE COM ECB ECB VARIETY ERS ERS SPT RST1ST 2ND X1069G 5 2 5 5 5 5 3563 5 3 6 3 4 5 36B08 5 6 5 6 5 35R57 4 3 55 4 4 35P12 5 3 5 6 5 5 34G81 4 3 6 5 4 4 34G13 6 2 6 5 5 5

Further Embodiments of the Invention

This invention includes hybrid maize seed of X1069G and the hybrid maizeplant produced therefrom. The foregoing was set forth by way of exampleand is not intended to limit the scope of the invention.

As used herein, the tern plant includes plant cells, plant protoplasts,plant cell tissue cultures from which maize plants can be regenerated,plant calli, plant clumps, and plant cells that are intact in plants, orparts of plants, such as embryos, pollen, ovules, flowers, kernels,ears, cobs, leaves, seeds, husks, stalks, roots, root tips, anthers,silk and the like.

Duncan, Williams, Zehr, and Widholm, Planta, (1985) 165:322-332 reflectsthat 97% of the plants cultured which produced callus were capable ofplant regeneration. Subsequent experiments with both inbreds and hybridsproduced 91% regenerable callus which produced plants. In a furtherstudy in 1988, Songstad, Duncan & Widholm in Plant Cell Reports (1988),7:262-265 reports several media additions which enhance regenerabilityof callus of two inbred lines. Other published reports also indicatedthat “nontraditional” tissues are capable of producing somaticembryogenesis and plant regeneration. K. P. Rao, et al., Maize GeneticsCooperation Newsletter, 60:6465 (1986), refers to somatic embryogenesisfrom glume callus cultures and B. V. Conger, et al., Pant Cell Reports,6:345347 (1987) indicates somatic embryogenesis from the tissue culturesof maize leaf segments. Thus, it is clear from the literature that thestate of the art is such that these methods of obtaining plants are, andwere, “conventional” in the sense that they are routinely used and haveartery high rate of success.

Tissue culture of maize is described in European Patent Application,publication 160,390, incorporated herein by reference. Maize tissueculture procedures are also described in Green and Rhodes, “PlantRegeneration in Tissue Culture of Maize,” Maize for Biological Research(Plant Molecular Biology Association, Charlottesville, Va. 1982, at367-372) and in Duncan, et al., “The Production of Callus Capable ofPlant Regeneration from Immature Embryos of Numerous Zea MaysGeneotypes,” 165 Planta 322-332 (1985). Thus, another aspect of thisinvention is to provide cells which upon growth and differentiationproduce maize plants having the genotype of X1069G.

Transformation of Maize

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional., or modified versions of native orendogenous genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreign,additional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transgenicversions of the claimed hybrid maize line X1069G.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used, alone or incombination with other plasmids, to provide transformed maize plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the maize plant(s).

Expression Vectors for Maize Transformation

Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e. inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (npfII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80: 4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5: 299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside 3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol. 8:1216 (1988), Jones et al., Mol. Gen. Genet., 210: 86 (1987), Svab etal., Plant Mol. Biol. 14: 197 (1990), Hille et al., Plant Mol Biol. 7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or broxynil. Comai et al.,Nature 317: 741-744 (1985), Gordon-Kamm et al., Plant Cell 2: 603-618(1990) and Stalker et al., Science 242: 419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5—eno/pyruvylshikimate3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13: 67(1987), Shah et al., Science 233: 478 (1986), Charest et al., Plant CellRep. 8: 643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include β-glucuronidase (GUS), β-galactosidase,luciferase and chloramphenicol acetyltransferase. Jefferson, R. A.,Plant Mol. Biol. Rep. 5: 387 (1987)., Teeri et al., EMBO J. 8: 343(1989), Koncz et al., Proc. Natl. Acad. Sci. U.S.A. 84:131 (1987), DeBlock et al., EMBO J. 3: 1681 (1984). Another approach to theidentification of relatively rare transformation events has been use ofa gene that encodes a dominant constitutive regulator of the Zea maysanthocyanin pigmentation pathway. Ludwig et al., Science 247: 449(1990).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes Publication 290 Imagene Green™, p. 1-4 (1993) and Naleway et al.,J. Cell Biol. 115: 151a. (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds, and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 26: 802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters

Genes included in expression vectors must be driven by a nucdeotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orscierenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissues arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression inmaize. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in maize. With an inducible promoter the rateof transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal. Plant Mol. Biol. 22: 361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al. PNAS 90: 4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen. Genetics 227: 229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243: 32-38 (1994)) or Tet repressor from Tn10 (Gatzet al., Mol. Gen. Genet. 227: 229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 8: 0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression inmaize or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in maize.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313: 810812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2: 163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol 12: 619-632 (1989) andChristensen et al., Plant Mol. Biol. 18: 675-689 (1992)): pEMU (Last etal., Theor. App. Genet 81: 581-588(1991)); MAS (Velten et al., EMBO J.3: 2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genet. 1: 276-285 (1992) and Atanassova et al., Plant Journal 2(3):291-300 (1992)).

The ALS promoter, a XbaVNcol fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence that has substantial sequencesimilarity to said XbaI/NcoI fragment), represents a particularly usefulconstitutive promoter. See PCT application W096/30530.

C. Tissue-specific or Tissue-preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin maize. Optionally, the tissue-specific promoter is operably linked toa nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in maize. Plants transformed with a geneof interest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferredpromoter,—such as that from the phaseolin gene (Murai et al., Science 2:476482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA82: 3320-3324 (1985)); a leaf-specific and light-induced promoter suchas that from cab or rubisco (Simpson et al., EMBO J. 4(11): 2723-2729(1985) and Timko et al., Nature 318: 579-582 (1985)); an anther-specificpromoter such as that from LAT53 Twell et a., Mol. Gen. Genet 217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol Gen. Genet. 224: 161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6: 217-224 (1993).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion, or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized. The presence of asignal sequence directs a polypeptide to either an intracellularorganelle or subcellular compartment or for secretion to the apoplast.Many signal sequences are known in the art. See, for example, Becker etal., Plant Mol. Biol. 20: 49 (1992), Close, P. S., Master's Thesis, IowaState University (1993), Knox, C., et al., “Structure and Organizationof Two Divergent Alpha-Amylase Genes From Barley”, Plant Mol. Biol. 9:3-17 (1987), Lemer et al., Plant Physiol. 91: 124-129 (1989), Fontes etal., Plant Cell 3: 483496 (1991), Matsuoka et al., Proc. Natl. Acad.Sci. 88: 834 (1991), Gould et al., J. Cell Biol 108: 1657 (1989),Creissen et al., Plant J. 2: 129 (1991), Kalderon, D., Robers, B.,Richardson, W., and Smith A., “A short amino acid sequence able tospecify nuclear location”, Cell 39: 499-509 (1984), Stiefel, V.,Ruiz-Avila, L., Raz R., Valles M., Gomez J., Pages M.,Martinez-Izquierdo J., Ludevid M., Landale J., Nelson T., andPuigdomenech P., “Expression of a maize cell wall hydroxyproline-richglycoprotein gene in early leaf and root vascular differentiation”,Plant Cell 2: 785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal Biochem. 114: 92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is maize. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RestrictionFragment Length Polymorphisms (RFLP), Polymerase Chain Reaction,(PCR)analysis, and Simple Sequence Repeats (SSR) which identifies theapproximate chromosomal location of the integrated DNA mnolecule. Forexemplary methodologies in this regard, see Glick and Thompson, METHODSIN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY 269-284 (CRC Press, BocaRaton, 1993). Map information concerning chromosomal location is usefulfor proprietary protection of a subject transgenic plant. Ifunauthorized propagation is undertaken and crosses made with othergermplasm, the map of the integration region can be compared to similarmaps for suspect plants, to determine if the latter have a commonparentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below.

1. Genes that Confer Resistance to Pests or Disease and that Encode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266: 789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladospodum fulvum);Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistanceto Pseudomonas sydngae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 7: 1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).(B) A Bacillus thudngiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene 48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection (Rockville, Md.), forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.(C) A lectin. See, for example, the disclosure by Van Damme et al.,Plant Molec. Biol. 24: 25 (1994), who disclose the nucleotide sequencesof several Clivia miniata mannose-binding lectin genes.(D) A vitamin-binding protein, such as avidin. See PCT applicationUS93/06487 the contents of which are hereby incorporated by. Theapplication teaches the use of avidin and avidin homologues aslarvicides against insect pests.(E) An enzyme Inhibitor, for example, a protease inhibitor or an amylaseinhibitor. See, for example, Abe et al., J. Biol. Chem. 262: 16793(1987) (nucleotide sequence of rice cysteine proteinase inhibitor), Huubet al., Plant Molec. Biol. 21: 985 (1993) (nucleotide sequence of cDNAencoding tobacco proteinase inhibitor 1), and Sumitani et al., Biosci.Biotech. Biochem. 57: 1243 (1993) (nucleotide sequence of Streptomycesnitrosporous α-amylase inhibitor).(F) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344: 458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.(G) An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269: 9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163: 1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.(H) An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116: 165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.(I) An enzyme responsible for an hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.(J) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achifnase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also: Kramer et al., InsectBiochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21: 673 (1993), who provide the nucleotide sequenceof the parsley ubl4-2. polyubiquitin gene.(K) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et a., Plant Molec. Biol. 24 (1994), of nucleotidesequences for mung bean calmodulin cDNA clones, and Griess et al., PlantPhysiol. 104: 1467 (1994), who provide the nucleotide sequence of amaize calmodulin cDNA clone.(L) A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.(M) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993),of heterologous expression of a cecropin-β lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.(N) A viratinvasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28: 451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus, Id.(O) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULARPLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).(P) A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 36: 469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.(Q) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10: 1436 (1992). The cloning and characterization of a gene whichencodes a bean endopolygalacturonase-inhibiting protein is described byToubart et al., Plant J. 2: 367 (1992).(R) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10: 305 (1992), have shown thattransgenic plants expressing the barley ribosome inactivabng gene havean increased resistance to fungal disease.

2. Genes that Confer Resistance to a Herbicide, For Example:

(A) A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7: 1241 (1988), and Miki et al., Theor. Appl. Genet. 80: 449(1990), respectively.(B) Glyphosate (resistance imparted by mutant 5enolpyruvl-3phosphikimatesynthase (EPSP) and aroA genes, respectively) and other phosphonocompounds such as glufosinate (phosphinothricin acetyl transferase (PAT)and Streptomyces hygroscopicus phosphinothricin acetyl transferase (bar)genes), and pyridinoxy or phenoxy proprionic acids and cycloshexones(ACCase inhibitor-encoding genes). See, for example, U.S. Pat. No.4,940,835 to Shah et al., which discloses the nucleotide sequence of aform of EPSP which can confer glyphosate resistance. A DNA moleculeencoding a mutant aroA gene can be obtained under ATCC accession No.39256, and the nucleotide sequence of the mutant gene is disclosed inU.S. Pat. No. 4,769,061 to Comai. European patent application No. 0 333033 to Kumada et al. and U.S. Pat. No. 4,975,374 to Goodman et al.disclose nucleotide sequences of glutamine synthetase genes which conferresistance to herbicides such as L-phosphinothricin. The nucleotidesequence of a phosphinothricin-acetyl-transferase gene is provided inEuropean application No. 0 242 246 to Leemans et al. De Greef et al.,Bio/Technology 7: 61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cycloshexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl Genet. 8: 435 (1992).(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrle (nitrilase gene). Przibilla et al.,Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J. 285:173 (1992).

3. Genes that Confer or Contribute to a Value-added Trait. Such As:

(A) Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearoyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.USA 89: 2624 (1992).

(B) Decreased Phytate Content

(1) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant. Forexample, see Van Hartingsveldt et al., Gene 127: 87 (1993), for adisclosure of the nucleotide sequence of an Aspergillus niger phytasegene.

(2) A gene could be introduced that reduces phytate content. In maize,this, for example, could be accomplished, by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid. See Raboyet al., Maydica 35: 383 (1990).

(C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteriol. 170: 810(1988) (nucleotide sequence of Streptococcus mutans fructosyltransferasegene), Steinmetz et al., Mol. Gen. Genet. 200: 220 (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al.,Bio/Technology 10: 292 (1992) (production of transgenic plants thatexpress Bacillus licheniformis α amylase), Elliot et al., Plant Molec.Biol. 21: 515 (1993) (nudeotide sequences of tomato invertase genes),Søgaard et al., J. Biol. Chem. 268: 22480 (1993) (site-directedmnutagenesis of barley α-amylase gene), and Fisher et al., PlantPhysiol. 102:1045 (1993) (maize endosperm starch branching enzyme II).Methods for Maize Transformation

Numerous methods W plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or issue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton,1993) pages 89-119.

A. Agrobacterium-mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227: 1229 (1985). A. tumefaciens and A.rhizogenes are plant pathogenic soil bacteria which geneticallytransform plant cells. The Ti and Ri plasmids of A. tumefaciens and A.rhizogenes, respectively, carry genes responsible for genetictransformation of the plant. See, for example, Kado, C. I., Crit. Rev.Plant. Sci. 10: 1 (1991). Descriptions of Agrobacterium vector systemsand methods for Agrobacterium-mediated gene transfer are provided byGruber et al., supra, Miki et al., supra, and Moloney et al., Plant CellReports 8: 238 (1989). See also, U.S. Pat. No. 5,591,616, issued Jan. 7,1997.

B. Direct Gene Transfer

Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice and maize. Hieiet al., The Plant Journal 6: 271-282 (1994); U.S. Pat. No. 5,591,616,issued Jan. 7, 1997. Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 Am. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. Technol. 5: 27 (1987), Sanford, J. C., Trends Biotech. 6: 299(1988), Klein et al., Bio/Technology 6: 559-563 (1988), Sanford, J. C.,Physiol Plant 79: 206 (1990), Klein et al., Biotechnology 10: 268(1992). In maize, several target tissues can be bombarded withDNA-coated microprojectiles in order to produce transgenic plants,including, for example, callus (Type I or Type II), immature embryos,and meristematic tissue.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9: 996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4: 2731 (1985), Chrlstouet a., Proc. Natl. Acad. Sci. U.S.A. 84: 3962 (1987). Direct uptake ofDNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol orpoly-L-omithine have also been reported. Hain et al., Mol. Gen. Genet.9: 161 (1985) and Draper et al., Plant Cell Physiol. 23: 451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4: 1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24: 51-61 (1994).

Following transformation of maize target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing transgenic inbred lines. Transgenic inbred lines could then becrossed, with another (non-transformed or transformed) inbred line, inorder to produce a transgenic hybrid maize plant. Alternatively, agenetic trait which has been engineered into a particular maize lineusing the foregoing transformation techniques could be moved intoanother line using traditional backcrossing techniques that are wellknown in the plant breeding arts. For example, a backcrossing approachcould be used to move an engineered trait from a public, non-elite lineinto an elite line, or from a hybrid maize plant containing a foreigngene in its genome into a line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

Industrial Applicability

Maize is used as human food, livestock feed, and as raw material inindustry. The food uses of maize, in addition to human consumption ofmaize kernels, include both products of dry- and wet-milling industries.

Maize, Including both grain and nonrain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs, and poultry.

Industrial uses of maize include production of ethanol, maize starch inthe wet-milling industry and maize flour in the dry-milling industry.The industrial applications of maize starch and flour are based onfunctional properties, such as viscosity, film formation, adhesiveproperties, and ability to suspend particles. The maize starch and flourhave application in the paper and textile industries. Other industrialuses include applications in adhesives, building materials, foundrybinders, laundry starches, explosives, oil-well muds, and other miningapplications.

Plant parts other than the grain of maize are also used in industry.Stalks and husks are made into paper and wallboard and cobs are used forfuel and to make charcoal.

The seed of the hybrid maize plant and various parts of the hybrid maizeplant and transgenic versions of the foregoing, can be utilized forhuman food, livestock feed, and as a raw material in industry.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that an certain changes andmodifications such as single gene modifications and mutations,somoclonal variants, variant individuals selected from large populationsof the plants of the instant hybrid may be practiced within the scope ofthe invention, as limited only by the scope of the appended claims.

DEPOSITS

Applicant(s) have made a deposit of at least 2500 seeds of hybrid maizeplant X1069G and inbred parent plants GE535769 and GE515721 with theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209 USA, ATCC Deposit Nos. PTA-5474, PTA-5522 andPTA-1306, respectively. The seeds deposited with the ATCC on Sep. 10,2003, Sep. 15, 2003 and Feb. 4, 2000, respectively were taken from thedeposit maintained by Pioneer Hi-Bred International, Inc., 800 CapitalSquare, 400 Locust Street, Des Moines, Iowa 50309-2340, since prior tothe filing date of this application. Access to this deposit will beavailable during the pendency of the application to the Commissioner ofPatents and Trademarks and persons determined by the Commissioner to beentitled thereto upon request. Upon allowance of any claims in theapplication, the Applicant(s) will make available to the public,pursuant to 37 C.F.R § 1.808, sample(s) of the deposit of at least 2500seeds of hybrid maize plant X1069G and inbred parent plants GE535762 andGE515721 with the American Type Culture Collection (ATCC), 10801Univerty Boulevard, Manassas, Va. 20110-2209. This deposit of seed ofhybrid maize plant X1069G and inbred parent plants GE535769 and GE515721will be maintained in the ATCC depository, which is a public depository,for a period of 30 years, or 5 years after the most recent request orfor the enforceable life of the patent, whichever is longer, and will bereplaced if it becomes nonviable during that period. Additionally,Applicant(s) have satisfied all the requirements of 37-C.F.R. §§1.801-1.909, including providing an indication of the viability of thesample upon deposit. Applicant(s) have no authority to waive anyrestrictions imposed by law on the transfer of biological material orits transportation in commerce. Applicant(s) do not waive anyinfringement of their rights granted under this patent or under thePlant Variety Protection Act (7 USC 2321 et seq.).

1. Seed of hybrid maize variety designated X1069G, representative seed of said variety having been deposited under ATCC Accession number PTA-5474.
 2. A maize plant, or a part thereof, produced by growing the seed of claim
 1. 3. Pollen of the plant of claim
 2. 4. An ovule of the plant of claim
 2. 5. A tissue culture of regenerable cells produced from the plant of claim
 2. 6. Protoplasts produced from the tissue culture of claim
 5. 7. The tissue culture of claim 5, wherein cells of the tissue culture are from a tissue selected from the group consisting of leaf, pollen, embryo, root, root tip, anther, silk, flower, kernel, ear, cob, husk and stalk.
 8. A maize plant regenerated from the tissue culture of claim 5, said plant having all the morphological and physiological characteristics of hybrid maize plant X1069G, representative seed of said plant having been deposited under ATCC Accession No. PTA-5474.
 9. A method for producing an F1 hybrid maize seed, comprising crossing the plant of claim 2 with a different maize plant and harvesting the resultant F1 hybrid maize seed.
 10. A maize plant, or part thereof, having all the physiological and morphological characteristics of the hybrid maize plant X1069G, representative seed of said plant having been deposited under ATCC Accession No. PTA-5474.
 11. A method of introducing a desired trait into a hybrid maize variety X1069G comprising: (a) crossing at least one of inbred maize parent plants GE535769 and GE515721, representative seed of which have been deposited under ATCC Accession Nos. PTA-5522 and PTA-1306 respectively, with another maize line that comprises a desired trait, to produce F1 progeny plants, wherein the desired trait is selected from the group consisting of male sterility, herbicide resistance, insect resistance, disease resistance and waxy starch; (b) selecting said F1 progeny plants that have the desired trait to produce selected F1 progeny plants; (c) backcrossing the selected progeny plants with said inbred maize parent plant to produce backcross progeny plants; (d) selecting for backcross progeny plants that have the desired trait and morphological and physiological characteristics of said inbred maize parent plant to produce selected backcross progeny plants; (e) repeating steps (c) and (d) three or more times in succession to produce a selected fourth or higher backcross progeny plant; and (f) crossing said fourth or higher backcross progeny plant with the other inbred maize parent plant to produce a hybrid maize variety X1069G with the desired trait and all of the morphological and physiological characteristics of hybrid maize variety X1069G listed in Table 1 as determined at the 5% significance level when grown in the same environmental conditions.
 12. A plant produced by the method of claim 11, wherein the plant has the desired trait and all of the physiological and morphological characteristics of hybrid maize variety X1069G listed in Table 1 as determined at the 5% significance level when grown in the same environmental conditions.
 13. The plant of claim 12 wherein the desired trait is herbicide resistance and the resistance is conferred to an herbicide selected from the group consisting of: imidazolinone, sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine and benzonitrile.
 14. The plant of claim 12 wherein the desired trait is insect resistance and the insect resistance is conferred by a transgene encoding a Bacillus thuringiensis endotoxin.
 15. The plant of claim 12 wherein the desired trait is male sterility and the trait is conferred by a cytoplasmic nucleic acid molecule that confers male sterility.
 16. A method of modifying fatty acid metabolism, phytic acid metabolism or carbohydrate metabolism in a hybrid maize variety X1069G comprising: (a) crossing at least one of inbred maize parent plants GE535769 and GE515721, representative seed of which have been deposited under ATCC Accession Nos. as PTA-5522 and PTA-1306 respectively, with another maize line that comprises a nucleic acid molecule encoding an enzyme selected from the group consisting of phytase, steryl-ACP desaturase, fructosyltransferase, levansucrase, alpha-amylase, invertase and starch branching enzyme; (b) selecting said F1 progeny plants that have said nucleic acid molecule to produce selected F1 progeny plants; (c) backcrossing the selected progeny plants with said inbred maize parent plant to produce backcross progeny plants; (d) selecting for backcross progeny plants that have said nucleic acid molecule and morphological and physiological characteristics of said inbred maize parent plant to produce selected backcross progeny plants; (e) repeating steps (c) and (d) three or more times in succession to produce a selected fourth or higher backcross progeny plant; and (f) crossing said fourth or higher backcross progeny plant with the other inbred maize parent plant to produce a hybrid maize variety X1069G that comprises said nucleic acid molecule and has all of the morphological and physiological characteristics of hybrid maize variety X1069G listed in Table 1 as determined at the 5% significance level when grown in the same environmental conditions.
 17. A plant produced by the method of claim 16, wherein the plant comprises the nucleic acid molecule and all of the physiological and morphological characteristics of hybrid maize variety X1069G listed in Table 1 as determined at the 5% significance level when grown in the same environmental conditions.
 18. A method for producing a maize seed, comprising crossing the plant of claim 2 with itself or a different maize plant and harvesting the resultant maize seed. 